English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using  Pichia pastoris  as an expression host.  P. pastoris  has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in  P. pastoris  using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A 2A R) in  P. pastoris  under control of a methanol-inducible promoter in both flask and bioreactor cultures.    Results  Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A 2A R, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in  P. pastoris . This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A 2A R, and therefore more suitable for further functional and structural studies.    Conclusion  Large-scale expression of the A 2A R in  P. pastoris  bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures